INTERNATIONAL JOURN~OF SYSTEMATIC BACTERIOLOGY,OCt. 1994, p. 659-664 Vol. 44, No. 4 0020-7713/94/$04.00 + 0 Copyright 0 1994, International Union of Microbiological Societies Description of Bacillus laevolacticus (ex Nakayarna and Yanoshi 1967) sp. nov., norn. rev. I. ANDERSCH,? S. PIANKA, D. FRITZE,* AND D. CLAUS DSM-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, 0-38124 Braunschweig, Germany The name “Bacillus laevolacticus” Nakayama and Yanoshi 1967 was not included on the Approved Lists of Bacterial Names and therefore has no standing in bacteriological nomenclature. In this study 22 catalase- positive, acid-tolerant, facultatively anaerobic, lactic acid-producingBacillus strains were examined taxonom- ically and compared with a number of strains belonging to phenetically similar Bacillus species (Bacillus coagulans, Bacillus smithii, “Bacillus vesiculiferous”) and with Sporolactobacillus. The G+ C contents (43 to 45 mol%), DNA-DNA homology values (72 to 98%), and results of phenetic similarity analyses revealed that the members of the 44B.laevolacticus” group were very homogeneous in their phenotypic and genotypic character- istics and clearly distinguishable from other Bacillus and SporolactobaciUus species. On the basis of these findings, revival of the name Bacillus laevolacticus is proposed. Traditionally, production of lactic acid is observed in micro- walls containing diaminopimelic acid were consistent with organisms which are grouped under the term “lactic acid the description of the genus Bacillus. Accordingly, these bacteria.” However, a considerable number of lactic acid- organisms were placed in a new genus, Sporolactobacillus, as producing, aerobic, spore-forming organisms have been de- Sporolactobacillus inulinus. Similar organisms, including scribed. These bacteria have been isolated from food or in “Sporolactobacillus laevas,” “Sporolactobacillus laevas var. connection with spoilage of preserved food, from milk (12,29), intermedius, ” and “Sporolactobacillus racemicus ,” have been from tomato puree (4), from the rhizospheres of various plants isolated from the rhizospheres of wild plants, but these (25), from a sugar production factory (18), and from the bacteria have not been validly described as new species (36, intestines of crayfish (34). It is possible that lactic acid produc- 37). tion is much more widely distributed among Bacillus species In 1967, Nakayama and Yanoshi (25) isolated and described than we realize at this time. The species of the genus Bacillus catalase-positive, acid-tolerant, facultatively anaerobic, meso- which have been reported to produce lactic acid include two recognized species, Bacillus coagulans and Bacillus smithii, and philic Bacillus strains that produced lactic acid, which they three species whose names have not been validly published named “B. laevolacticus” and “B. racemilacticus.” These organ- previously, “Bacillus laevolacticus,” “Bacillus racemilacticus,” isms could be distinguished from each other only by the ability and “Bacillus vesiculi$erous.” of “B. racemilacticus” to grow in the presence of 3.5% NaCl B. coagulans was first isolated by Hammer in 1915 from and to produce m-lactic acid instead of D-(-)-lactic acid [“B. spoiled canned milk and was described as a new species. In a laevolacticus” produced D-( -)-lactic acid]. “B. laevolacticus” number of later studies it was noted that the cell morphology, and “B. racemila~ticus’~were distinguished from B. coagulans spore surface morphology, and sporangium morphology varied on the basis of their lower growth temperatures, greater acid from strain to strain. This high degree of variability led to the tolerance, requirement of carbohydrates for growth, and ste- creation of a number of other species names which later were reospecificity of the lactate produced [B. coagulans produces recognized as subjective synonyms, including “Bacillus ther- only L-(+)-lactic acid from glucose]. As early as 1981, Collins moacidurans” (5), “Bacillus thermoacidificans” (28), “Bacillus and Jones (9) stated that, on the basis of chemotaxonomic dextrolacticus” (l),and “Lactobacillus cereale” (26). Later, it data, the acid-tolerant Bacillus strains (namely “B. laevolacti- was observed that clustering of strains was obtained when cus,” “B. racemilacticus,” “Bacillus myxo1acticus,” and “Bacil- certain physiological tests were performed (18, 21, 35). Naka- lus dextrolacticus”) and Sporolactobacillus strains could be mura et al. (24) distinguished DNA relatedness groups, of grouped together as members of a separate taxon close to the which DNA group 1 was identified to represent the species B. genus Bacillus. Yanagida et al. (36, 37) included a number of coagulans sensu stricto. DNA group 2 was described as a new “B. laevolacticus” and “B. racemilacticus” strains in their species, which was named B. smithii. studies. Certain strains clustered together, but clear separation Other bacterial strains that share some of the characteristics from Sporolactobacillus strains was not possible. On the basis of the genera Lactobacillus and Bacillus were isolated from chicken feed by Kitahara and Suzuki (17). These strains were of the results of 16s rRNA sequencing studies performed with similar to members of the genus Lactobacillus in their lack of a set of these strains, Suzuki and Yamasoto (32) found that catalase, microaerophilic growth, and lactic acid fermentation most of them clustered more or less around the genus Sporo- characteristics, but production of typical endospores and cell lactobacillus, forming several subclusters, while a single strain of “B. racemilacticus” formed a separate branch and one strain clustered in Bacillus rRNA group 1 (2). In order to clarify the taxonomic position of 22 catalase- * Corresponding author. Mailing address: DSM-Deutsche Sammlung positive, acid-tolerant, facultatively anaerobic, mesophilic Ba- von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg lb, cillus strains with respect to a number of previously recognized D-38124 Braunschweig, Germany. Phone: 49-531-2616-254. Fax: 49- 53 1-2616-4 18. Bacillus species, we examined the phenotypic and chemosys- Present address: Bayer AG, Pflanzenschutzzentrum Monheim, tematic characteristics, DNA base compositions, and levels of D-51368 Leverkusen, Germany. DNA relatedness of these organisms. 659 660 ANDERSCH ET AL. INT.J. SYST.BACTERIOL. TABLE 1. Acid-tolerant Bacillus strains examined in this study Strain Received as: Source” Strain history and original designation“ Other designation(s) DSM 442T M ST 1 <O. Nakayama, “B. laevolacticus,” rhizosphere of NCIMB 10269T,ATCC 23492T, JCM 2513T Ranunculus sceleratus DSM 444’ M 14 1 <O. Nakayama, “B. racemilacticus,” rhizosphere of NCIMB 10274, ATCC 23496, JCM 2517 Lactuca dentata DSM 2310 NCIMB 10270 2 <NCIMB < 0.Nakayama, M 1, “B. laevolacticus,” ATCC 23493, JCM 2514 rhizosphere of Trifolium repens DSM 2315‘ NCIMB 10276 2 <NCIMB < 0.Nakayama, M 64, “B. racemilacticus,” rhizosphere DSM 2316 NCIMB 10271 2 <NCIMB < 0. Nakayama, M 7, “B. laevolacticus,” ATCC 23494, JCM 2515 rhizosphere of Allium japonicum DSM 2317 NCIMB 10272 2 <NCIMB < 0.Nakayama, M 40, “B. ATCC 23495 laevolacticus,” rhizosphere DSM 2318 NCIMB 10273 2 <NCIMB < 0. Nakayama, M 4, “B. laevolacticus,” ATCC 23549, JCM 2516 rhizosphere of Hemerocallis jluva DSM 6474 IAM 12329 3 <IAM < 0. Nakayama, M 71, “B. laevolacticus,” rhizosphere DSM 6475 IAM 12331 3 <IAM < 0. Nakayama, M 104, “B. laevolacticus,” rhizosphere DSM 6476 IAM 12326 3 <IAM < 0. Nakayama, M 105, “B. laevolacticus,” rhizosphere DSM 6477 M 81 1 <O. Nakayama, “B. laevolacticus,” rhizosphere DSM 6478 IAM 12379 3 <IAM < 0. Nakayama, M 75, “B. laevolacticus,” rhizosphere DSM 6510 M 89 1 <O. Nakayama, “B. laevolacticus,” rhizosphere DSM 6511 M 121 1 <O. Nakayama, “B. laevolacticus,” rhizosphere DSM 6547 IAM 12327 3 <IAM < 0. Nakayama, M 66, “B. laevolacticus,” rhizosphere DSM 6548 IAM 12328 3 <IAM < 0. Nakayama, M 68, “B. laevolacticus,” rhizosphere DSM 6549 IAM 12330 3 <IAM < 0.Nakayama, M 91, “B. laevolacticus,” rhizosphere DSM 6763 M 120 1 <O. Nakayama, “B. laevolacticus,” rhizosphere DSM 6764 M 95 1 <O. Nakayama, “B. laevolacticus,” rhizosphere DSM 6771 M 100 1 <O. Nakayama, “B. laevolacticus,” rhizosphere DSM 2309 NCIMB 10275 2 <NCIMB < 0. Nakayama, M 5, “B. ATCC 23497, JCM 2518 racemilacticus,” rhizosphere of Rumex acetosa DSM 2314d NCIMB 10276 2 <NCIMB < 0. Nakayama, M 39, “B. ATCC 23498 racemilacticus,” rhizosphere 1, 0. Nakayama, Yamanashi University, Kofu, Japan; 2, National Collections of Industrial and Marine Bacteria, Ltd. (NCIMB), Aberdeen, Scotland; 3, Institute of Applied Microbiology (IAM), University of Tokyo, Tokyo, Japan. Designated the type strain of “B. racemilacticus” by Gibson and Gordon (10) but identified as a “B. laevolacticus” strain by Blumenstock (6). Identified as a “B. racemilacticus” strain by Nakayama (24a) and as a “B. laevolacticus” strain by Blumenstock (6). Identified as a “B. racemilacticus” strain by Nakayama and Yanoshi (25) and as a B. coagulans strain by Blumenstock (6). MA.TERIALS AND METHODS Nakayama and Yanoshi (25). For the nitrate reduction and indole formation tests the medium was supplemented with 1% Bacterial strains. The Bacillus strains used in this study are (final concentration) glucose. listed in Table 1. Seven strains were obtained from the The following tests were performed by previously described National Collections of Industrial and Marine Bacteria, Ltd., methods: flagellum staining (13), Gram staining (3), oxidase Aberdeen,